1. Field of the Invention
The present invention relates to the use of alkylaryl polyether alcohol polymers in the treatment of chronic inflammation. More particularly, the present invention relates to the use of alkylaryl polyether alcohol polymers to reduce the activation of nuclear factor kappa B (NF-.kappa.B) and inhibit the secretion of pro-inflammatory cytokines TNF-alpha (TNF-.alpha.), interleukin-1 beta (IL-1.beta.), interleukin-6 (IL-6), interleukin-8 (IL-8) and the growth factor granulocyte macrophage colony stimulating factor (GM-CSF).
2. The Prior Art
Discussion of oxidant-mediated injury.
Oxygen is life-giving to aerobic plants and animals who depend on it for energy metabolism. It can also be lethal to those same organisms when it is altered from its stable dioxygen (O.sub.2) state to any one of three partially reduced species: a) the one electron reduced form superoxide anion (.sup..cndot. O.sub.2.sup.-); the two electron reduced form hydrogen peroxide (H.sub.2 O.sub.2); or the deadly three electron reduced form the hydroxyl radical (.sup..cndot. OH). In biologic systems O.sub.2- and H.sub.2 O.sub.2 are metabolic byproducts of a host of enzymes (oxygenases) that use oxygen as a cofactor. H.sub.2 O.sub.2 is also produced from .sup..cndot. O.sub.2.spsb.- by the enzymatic action of superoxide dismutases. However, .sup..cndot. OH is generally produced only when .sup..cndot. O.sub.2.spsb.- and H.sub.2 O.sub.2 interact with transitional ions of metals such as iron, copper, nickel or vanadium in dangerous cyclical redox reactions: ##STR2## The above reactions are termed the superoxide-driven Fenton reaction common in biological systems. The Fenton reaction can also be initiated by other reducing substances such as ascorbate in the presence of ferric iron and H.sub.2 O.sub.2.
While .sup..cndot. O.sub.2.spsb.- and H.sub.2 O.sub.2 are each toxic for biological systems, .sup..cndot. OH (and its alternant hypothesized form the ferryl intermediate FeO.sup.2+) is a highly reactive species that can oxidize unsaturated membrane lipids, damage cellular proteins and cause mutagenic strand breaks in DNA. To prevent injury from partially reduced O.sub.2 species under normal conditions, cells have evolved an elaborate system of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and antioxidant molecules (gluathione, alpha-tocopherol, beta carotene). However, when production of partially reduced O.sub.2 species exceeds capacity of antioxidant defenses to contain them, oxidant injury occurs.
A growing number of mammalian disease entities are now thought to be related to overproduction of partially reduced O.sub.2 species, including the reperfusion injury syndromes myocardial infarction and stroke, adult respiratory distress syndrome, oxygen toxicity of the lung, lung injury from asbestos, Parkinson's disease, thermal and solar burns of the skin, injury to the gastrointestinal tract from nonsteroidal anti-inflammatory agents and airway diseases such as chronic bronchitis, asthma and cystic fibrosis (see Table IV, page 60, B. Halliwell and J. M. C. Gutteridge. Methods in Enzymology (1990) 186:1-85). Treatment of these conditions is increasingly directed either toward strategies that prevent enzymatic production of partially reduced O.sub.2 species and to the introduction of exogenous antioxidant compounds that restore oxidant-antioxidant balance in biologic and chemical systems. More recently, as will be outlined below, treatment of inflammation in many of these conditions has been directed toward interrupting activation of the transcription factors mediating the genetic expression of pro-inflammatory cytokines important in the pathogenesis of these conditions.
Discussion of transcription factors and cytokines.
Transcription factors are cellular proteins that bind to regulatory sequences of genes and increase or decrease the rate of gene transcription. By affecting the rate of gene transcription, transcription factors play a critical role in regulation of cell function during health and disease. Among the most important transcription factors in disease are those that regulate expression of the genes for pro-inflammatory cytokines. These cytokines are secreted cellular proteins that dramatically affect the behavior of other cells. As examples, the cytokine TNF-.alpha. causes weight loss in patients with tumors or chronic infections, produces cellular death and is thought to be an important mediator of septic shock. The cytokine IL-1.beta. mediates fever, and shares many of the properties of TNF. The cytokine IL-8 (and its close relatives such as RANTES) is a potent chemotactic signal aiding in the recruitment of inflammatory cells such as neutrophils. The cytokine GM-CSF signals the bone marrow to produce more inflammatory cells, activates those cells once produced and lengthens their survival.
These cytokines play important roles in mediating the pathogenesis of such inflammatory diseases as cystic fibrosis, chronic bronchitis, asthma and viral infections, among many others (T. L. Bonfield, et al. "Inflammatory cytokines in cystic fibrosis lungs". American Journal of Respiratory and Critical Care Medicine (1996) In Press; N. G. McElvaney, et al. "Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor". Journal of Clinical Investigation (1992) 90:1296-1301; K. D. Pfeffer, et al. "Expression and regulation of tumor necrosis factor in macrophages from cystic fibrosis patients". American Journal of Respiratory, Cell and Molecular Biology (1993) 9:511-519; G. Williams and B. P. Giroir. "Regulation of cytokine gene expression: Tumor necrosis factor, interleukin-1, and the emerging biology of cytokine receptors". New Horizons (1995) 3:276-287; C. A. Dinarello. "Role of interleukin-1 and tumor necrosis factor in systemic responses to infection and inflammation". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 211-232; W. C. Greene. "The interleukins". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 233-245; M. Baggiolini, et al. "Interleukin-8 and related chemotactic cytokines". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 247-263; D. W. Golde and G. C. Baldwin. "Myeloid growth factors". In Inflammation: Basic Principles and Clinical Correlates, second edition. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, Ltd., New York (1992) p. 291-301; R. J. Horwitz and W. W. Busse. "Inflammation and asthma". Clinics in Chest Medicine (1995) 16:583-602).
These cytokines share regulation of their expression by the transcription factor Nuclear Factor kappa-B (NF-.kappa.B), a particularly important transcription factor mediating inflammatory events (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Biology (1994) 10:405-455). NF-.kappa.B is also an important transcriptional regulator of chemokines such as RANTES (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Biology (1994) 10:405-455) and of inducible nitric oxide synthase (iNOS) (P. J. Nelson, et al. "Genomic organisation and transcriptional regulation of the RANTES chemokine gene". Journal of Immunology (1993) 151:2601-2612), the enzyme producing nitric oxide (NO.sup..cndot.), a critical oxidant chemical produced as part of the pathogenesis of septic shock. NF-.kappa.B is present in the cytoplasm in an inactive form complexed to an inhibitory protein I.kappa.B. A number of events, yet to be completely characterized, cause I.kappa.B to dissociate from NF-.kappa.B in the cytoplasm. Free NF-.kappa.B then localizes to the nucleus, where it binds to a specific .kappa.B recognition site in the promoter region of target genes, prompting their expression. NF-.kappa.B is activated by a number of stimuli, including cytokines themselves, and by lipopolysaccharide (LPS) (U. Siebenlist, G. Granzuso and R. Brown. "Structure, regulation and function of NF-.kappa.B". Annual Review of Cell Bioloqy (1994) 10:405-455). NF-.kappa.B is also activated by oxidants such as hydrogen peroxide (M. Meyer, R. Schreck, and P. A. Baeverie. "H.sub.2 O.sub.2 and antioxidants have opposite effects on the activation of NF-.kappa.B and AP-1 in intact cells: AP-1 as secondary antioxidant response factor". EMBO Journal (1993) 12:2005-2015), suggesting that it may be an oxidant-stress responsive transcription factor. Conversely, some of the most potent inhibitors of NF-.kappa.B activation are compounds which can also act as antioxidants. A few, but not most, antioxidants prevent activation of NF-.kappa.B by LPS, prevent increases in corresponding messenger RNAs for inflammatory cytokines and decrease levels of TNF and IL-1 in the circulation following LPS injection (E. M. Eugui, et al. "Some antioxidants inhibit, in a co-ordinate fashion, the production of tumor necrosis factor .alpha., IL-1.beta. and IL-6 by human peripheral blood mononuclear cells". International Journal of Immunology (1993) 6:409-422; R. Schreck, et al. "Dithiocarbamates as potent inhibitors of nuclear factor .kappa.B activation in intact cells". Journal of Experimental Medicine (1992) 175:1181-1194). However, the few antioxidants known to inhibit NF-.kappa.B activation share no common structural similarity distinguishing them from those antioxidants that fail to prevent activation of NF-.kappa.B (see Eugui, above), preventing one skilled in the art from predicting which antioxidant compounds will and which will not favorably reduce NF-.kappa.B activation as a strategy of ameliorating inflammatory events in disease.
Another class of compounds known to inhibit NF-.kappa.B activation are anti-inflammatory steroids. Steroids act by combining in the cytoplasm with an intracellular protein called the Glucocorticoid Receptor (GR). Previously, the anti-inflammatory action of steroids was thought to occur exclusively as a result of passage of the GR-steroid complex to the nucleus, where the complex attaches to and influences regulatory gene regions called Glucocorticoid Responsive Elements (GREs). However, recently it has been shown that a major mechanism of anti-inflammatory steroid activity is inhibition of NF-.kappa.B (I. M. Adcock, et al. "Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells". American Journal of Physiology (1995) 268(Cell Physiology 37):C.sub.331 -C.sub.338). The GR-steroid complex prevents activation of NF-.kappa.B by directly interacting with free NF-.kappa.B in the cytoplasm, preventing NF-.kappa.B from translocating to the nucleus (A. Ray and K. E. Prefontaine. "Physical association and functional antagonism between the p65 subunit of transcription factor NF-.kappa.B and the glucocorticoid receptor". Proceedings of the National Academy of Sciences, USA (1994) 91:752-756). However, the GR-steroid complex accomplishes inhibition of NF-.kappa.B by mutual repression. By combining with free NF-.kappa.B in the cytoplasm, it too is kept from translocating to the nucleus to up-regulate other anti-inflammatory events. Indeed, mutual repression is thought to explain in part the phenomenon of steroid resistance in severe asthmatics. IL-1, IL-6, TNF and other pro-inflammatory cytokines secreted in the airway during an asthma attack increase cellular activation of NF-.kappa.B, providing more NF-.kappa.B subunits to bind GR-steroid complexes, reducing the amount of GR-steroid complex available to translocate to the nucleus (P. J. Barnes, A. P. Greening and G. K. Crompton. "Glucocorticoid resistance in asthma". American Journal of Respiratory and Critical Care Medicine (1995) 152:S125-S142).
Discussion of alkylaryl polyether alcohol polymers, including tyloxapol.
Antioxidants are compounds that can be easily oxidized to stable chemical forms. They can protect chemical and biologic systems by sacrificing themselves to oxidation in preference to oxidation of critically important chemical and biological molecules. Not all oxidizable compounds can perform antioxidant function. To successfully protect chemical and biologic systems from oxidants, the antioxidant must have a higher reactivity for the oxidant than the chemical or biologic molecule which it seeks to protect. To protect the desired chemical and biologic system from oxidation, it is also necessary for the antioxidant to partition itself adjacent to the molecule to be protected. As an example, a molecule to be protected within the lipid bilayer of plasma, endosomal or nuclear membranes might be best protected by an antioxidant with, at least in part, a lipophilic structure, so that it is partitioned to or near the lipid portion of the membrane, adjacent to the molecule needing protection from oxidation.
We have recently shown that a previously known class of drugs, the alkylaryl polyether alcohol polymers, are potent antioxidants useful in the treatment of mammalian diseases (U.S. Pat. No. 5,474,760 issued Dec. 12, 1995 and U.S. Ser. No. 08/039,732 filed Mar. 30, 1993, now abandoned, both to Ghio, Kennedy and Piantadosi). Alkylaryl polyether alcohol polymers are used commercially as surface active detergents and wetting agents (U.S. Pat. No. 2,454,541 to Bock and Rainey). The best known of this class is tyloxapol, a polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with formaldehyde and oxirane. However, other compounds in the class, sharing the properties of tyloxapol, are well known in the art (J. W. Cornforth, et al. "Antituberculous effect of certain surface-active polyoxyethylene ethers in mice". Nature (1951) 168:150-153).
Tyloxapol is relatively nontoxic and does not hemolyze red blood cells in a thousand times the concentrations at which other detergents are hemolytic (H. N. Glassman. "Hemolytic activity of some nonionic surface-active agents". Science (1950)111:688-689). Tyloxapol has been used in human pharmacologic formulations for over 30 years (M. L. Tainter, et al. "Alevaire as a mucolytic agent". New England Journal of Medicine (1955) 253:764-767). For instance, a composition sold by Winthrop Laboratories (a division of Sterling Drug, Inc.) and by Breon Laboratories (subsidiary of Sterling Drug, Inc.) under the trademark "ALEVAIRE", containing 0.125% "SUPERINONE" (brand of tyloxapol) in combination with 2% sodium bicarbonate and 5% glycerin, had been marketed for about 30 years for treatment of mucous secretions in patients with diseases and disorders such as chronic bronchitis, croup, pertussis, and poliomyelitis. (See, for example, a product brochure entitled "ALEVAIRE" Detergent Aerosol for Inhalation" (November, 1961) distributed by Breon Laboratories.).
However, in December of 1981, "ALEVAIRE" was withdrawn by the Food and Drug Administration for lack of efficacy for treatment of mucous secretions in patients with diseases and disorders such as chronic bronchitis, croup, pertussis, and poliomyelitis because it was found that there was no evidence that the tyloxapol in "ALEVAIRE" had any effect on secretions in the lung from diseases such as chronic bronchitis other than that of water in thinning secretions by simple dilution, and that papers in the manufacturer's bibliography were based on clinical impression and did not reflect adequate controls. (See, letter dated May 27, 1994 to Dr. Thomas Kennedy, one of the co-inventors of the present application, from Ms. Carolann W. Hooton, Chief, Freedom of Information Office, Center for Drug Evaluation and Research, Department of Health & Human Services, Public Health Service, Food and Drug Administration, Rockville, Md.).
Surprisingly, the present inventors have found that alkylaryl polyether alcohol polymers of the class typified by tyloxapol, are potent inhibitors of the activation of NF-.kappa.B, thereby preventing cellular production of pro-inflammatory cytokines.
Synopsis of background discussion.
Inflammation in a multitude of diseases is mediated by activation of the transcription factor NF-.kappa.B, which in turn causes an increase in cellular production of pro-inflammatory cytokines such as TNF, IL-1, IL-6, IL-8 and the growth factor GM-CSF, and an increase in critical cellular enzymes, such as inducible nitric oxide synthase (iNOS). The current treatment available to prevent activation of NF-.kappa.B and subsequent cytokine secretion is anti-inflammatory glucocorticoids. Recently a few, but not most, antioxidants have been found to also inhibit NF-.kappa.B.
It is theoretically possible to synthesize a multitude of compounds with antioxidant properties. However, there is no predictable structural similarity among the few agents shown to prevent NF-.kappa.B activation. Thus, the demonstration that a compound shows antioxidant activity would not, in of itself, predict that the same compound would also inhibit NF-.kappa.B activation and secretion of pro-inflammatory cytokines. Also, the factor limiting use of antioxidants as treatments in biologic systems is the inherent toxicity of many antioxidant compound themselves. Likewise, anti-inflammatory cortosteroids are potent inhibitors of NF-.kappa.B, but their use as such is severely limited by the well-known side effects of corticosteroids, including glucose intolerance, hypertension, bone resorption, weight gain and cataracts. Thus, it is a major advantage to discover that a class of commonly used and nontoxic ingredients in medicinal pharmacologic preparations are not only potent antioxidants, but also potent inhibitors of NF-.kappa.B activation. Not only can such compounds be used as treatments for diseases where antioxidants might be predicted to be of value, but they can be used as treatments for NF-.kappa.B mediated inflammatory conditions without themselves causing toxicity to biologic systems.